Abstract
The main psychoactive ingredient in the plant Cannabis sativa is Δ9-trans-tetrahydrocannabinol, which was found to signal through the cannabinoid CB1 receptor (CB1). CB1 is one of two main G protein-coupled receptors (GPCRs) in the endocannabinoid system. Though CB1 has been shown to preferentially couple to Gαi/o proteins, there has been evidence for promiscuous coupling to other classes. Gα12/13 proteins are one of the four main families of G proteins and act through Rho-guanine nucleotide exchange factors to stimulate RhoA. This leads to the activation of Rho-associated kinase and regulation of the actomyosin cytoskeleton. To modulate the signalling of GPCRs, arrestins have been demonstrated to have a key role in governing the processes of internalisation and desensitisation. Here, this research aimed to investigate the role of Gα12/13 proteins in the modulation of CB1-mediated β-arrestin recruitment. In HEK293A wild type (WT) cells, CB1 activated both Gα12 and Gα13 proteins in a concentration-dependent manner in TRUPATH G protein dissociation bioluminescence resonance energy transfer (BRET) assays. CB1-mediated β-arrestin 2 recruitment was enhanced in a concentration-dependent manner in HEK293A Gα12/13 protein knock-out (ΔGα12/13) cells, compared to WT cells in BRET β-arrestin translocation assays. β-arrestin 2 recruitment was almost abolished with the reintroduction of Gα12/13 proteins. Phosphorylation of extracellular-signal regulated kinase was assessed using a BRET biosensor assay, showing attenuated levels in ΔGα12/13 cells compared to WT cells, which was not restored when adding back Gα12/13 proteins. Receptor trafficking revealed no differences in cell surface half-life of CB1 between WT and ΔGα12/13 cells, though the addition of β-arrestin reduced half-life in both cell lines. Interestingly, the morphology of ΔGα12/13 cells appeared to have more cytoskeletal outgrowths than WT cells, which was reversed when Gα12/13 proteins were reintroduced. Overall, we demonstrate that ΔGα12/13 proteins are activated at CB1, though their modulatory role on CB1-mediated β-arrestin recruitment requires further study.